Deoxyribonucleic acid (DNA), and ribonucleic acid (RNA), polymers of nucleic acids arranged in a particular order which contain inheritable genetic information, are employed in a wide variety of research, medical, diagnostic and industrial processes. The variety of uses for extracted and purified DNA and RNA from disparate sources is rapidly increasing with the advent of biotechnology as an industry for the production of pharmaceutical, agricultural, pesticidal and other agents. Additionally nucleic acid sequences are being increasingly employed for their ability to detect and identify genetic and familial disorders and carrier states; genetic aberrations found in tumors; and proof of identity or parentage. Nucleic acid sequences are also being employed to aid in the detection of infections by bacteria, viruses and other agents.
The production of genetically engineered proteins and polypeptides is another area where purified nucleic acid sequences are in demand. DNA and RNA are employed as starting materials in the manufacture of a variety of products, including nucleoside antibiotic and antiviral agents. DNA and RNA libraries and clones selected from them are also routinely employed in molecular biology and biotechnological research.
Generally nucleic acid sequences must be extracted from biological sources, e.g., tissue samples, bacteria, viruses, salmon sperm and the like, and purified by separation from proteins, salts and other biological molecules prior to use.
The standard procedure for isolating DNA from a cell source involves digestion with a combination of a proteolytic enzyme and a non-ionic or anionic detergent, such as sarcosyl or sodium dodecyl sulfate. The resulting digest is extracted with a mixture of phenol and chloroform, which removes most of the hydrolyzed products. The DNA is then precipitated from the resulting aqueous phase by the addition of alcohol.
For example, genomic DNA is extracted from eukaryotic cells by incubating them with a proteolytic enzyme (usually proteinase k) and the anionic detergent, SDS (sodium dodecyl sulfate). The resulting mixture is extracted with a mixture of phenol and chloroform, which leaves the DNA and RNA in the aqueous phase. The DNA is then precipitated by the addition of ethanol and sodium acetate, and resuspended in a buffer. RNase is then used to hydrolyse the RNA, and the DNA is collected by phenol/chloroform extraction and ethanol precipitation. Alternatively, cell nuclei can be prepared by dounce homogenization (or frozen pulverisation), filtration and centrifugation through sucrose. The nuclei are treated with proteinase K and SDS as above. Preparations of DNA should not contain impurities which inhibit the enzymes used to manipulate it further, such as restriction endonucleases or the Tag polymerase for use in the polymerase chain reaction. For some applications, the DNA should be very long. In these cases, DNA is prepared by the same general methods, but applied to cells imbedded in an agarose gel.
DNA in plasmids exists as double-stranded, closed circular DNA in the host bacteria. It is harvested by lysing the bacteria with lysozyme and a nonionic detergent. This liberates the plasmids, but leaves most of the bacterial DNA adherent to the cell debris, which can be removed by ultra-centrifugation. The plasmid is then separated by density-gradient ultra-centrifugation in the presence of ethidium bromide. Alternatively, the bacteria are treated with sodium hydroxide, and the plasmid DNA is separated by exclusion gel chromatography after RNase treatment.
DNA from bacteriophages or other viruses can be collected (after removing contaminating bacterial DNA with a DNase) by precipitation with polethylene glycol followed by extraction with chloroform/phenol and ethanol precipitation.
DNA is usually purified from reaction mixtures by chloroform/phenol extraction and ethanol precipitation. Spermine can be used to precipitate DNA, and can subsequently be removed by dialysis. DNA can be harvested by its binding to powdered glass in the presence of high salt solutions, or by adsorption and elution from commercially prepared columns.
The extraction of full length RNA requires methods that inhibit RNase's, which are ubiquitous. Cells can be ruptured by the addition of SDS in the presence of an RNase inhibitor, and extracted with phenol/chloroform followed by ethanol precipitation. Alternatively, cells can be lysed by the addition of guanidinium isothiocyanate followed by ultra-centrifugation through a cesium chloride gradient.
RNA is extracted by a variety of techniques designed to protect it from the action of RNAase's. One conventional method employs the chaptropic agent, guanidinium isothiocyanate, followed by ultracentrifugation to harvest the dense RNA. Another standard method for obtaining RNA from cell sources uses hot phenol extraction, followed by digestion of DNA with DNAase, extraction with phenol/chloroform, and precipitation with alcohol. In the intermediate steps of the processing of DNA, phenol/chloroform extraction and ethanol precipitation are often used.
The above-described nucleic acid extraction and purification techniques are described in detail in a number of standard molecular biology methodological texts, including T. Maniatis et al., "Molecular Cloning. A Laboratory Manual.", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982) and L. G. Davis et al, "Basic Methods in Molecular Biology", Elsevier, N.Y. (1986).
A number of cationic detergents have been shown to be able to precipitate DNA and RNA from aqueous phases. Cationic detergents consist of a positively charged head group, which is usually a quaternary amine or a pyridinium group, and an aliphatic tail. Examples of commercially useful detergents include cetyl pyridinium bromide, cetyltrimethylammonium bromide (collectively known as cetrimonium compounds) and alkylbenzyldimethylammonium chlorides (collectively known as benzalkonium compounds). As the length of the side chain is increased, the resulting detergent become stronger, and its solubility in water decreases. Typical cationic detergents used in these procedures are cetyl pyridinium bromide, and cetyl trimethylammonium bromide, among others. See, e.g., A. S. Jones, Nature, 199:280-82 (1963); J. H. Weil and J. P. Ebel, Biochem. Biophys. Acta., 55:836-840 (1962). The ability of cationic detergents to precipitate DNA and RNA was reported by A. S. Jones, Biochem. Biophys. Acta.. 10:607 (1953). In a typical application, micro-organisms were extracted with phenol-p-aminosalicylate, and the DNA in the extract was precipitated with ethanol. The precipitate was dissolved in IM NaCl (in which cytoplasmic RNA is insoluble), and the DNA was precipitated by the addition of cetyl trimethylammonium bromide (CTAB), after dilution to 0.55 to 0.60M NaCl. The DNA/CTAB complex was redissolved in 1M NaCl and reprecipitated with ethanol. The solubility of RNA and DNA complexes with cationic detergents in polar organic solvents was reported by French researchers. These cationic detergents included cetrimonium compounds and benzalkonium bromides, and the solvents included ethanol and formamide. These workers showed that the nucleotides could be precipitated from the organic solvent by the addition of sodium chloride. The ability of benzalkonium compounds to solubilise proteins at the same time as precipitating nucleotides was not mentioned. Other workers have reported similar findings.
There remains a need in the art for additional, simpler and more efficient methods for extraction and purifying nucleic acids from cell sources.